Coronaviruses (CoVs) are a family of positive-stranded RNA viruses that cause respiratory disease in humans ranging in severity from the common cold to severe acute respiratory syndrome (SARS). The continued emergence of CoVs into the human population, the presence of CoVs within multiple animal species, and the identification of numerous SARS-CoV-like viruses in bats, indicates that there is likely a worldwide reservoir of CoVs that are currently and constantly replicating and evolving. Exactly how coronaviruses, which contain the largest known RNA genomes, balance maintaining their genomic stability with acquiring the diversity required for their demonstrated trans-species movement remains unknown. The current paradigm is that the evolution of RNA viruses is primarily a product of error-prone viral RNA-dependent RNA polymerases (RdRps), due to the absence of error recognition and repair (proofreading) activities during replication. Our work challenges this paradigm, and provides the first demonstration of an RNA virus-encoded protein (nsp14-ExoN), distinct from the RdRp, that modulates viral replication fidelity. Thus, defining the viral protein determinants of replication fidelity, and how they work together, will be critical in understanding coronavirus trans-species movement, and in the development of novel antiviral therapeutics targeting these viral proteins. The overall goal of this proposed research project is to identify viral protein determinants of fidelity, and to define their role in maintaining high-fielity replication. In Specific Aim 1, we will determine the role of nonstructural protein 10 (nsp10) in the regulation of nsp14-ExoN-mediated coronavirus replication fidelity in culture. We will engineer mutations predicted to alter nsp10-nsp14 interactions, and use deep sequencing and nucleoside analogs to determine the role of these mutations on coronavirus fidelity. In Specific Aim 2, we will define fidelity determinants outside of nsp14-ExoN and determine their role in maintaining high-fidelity replication. We will use a combination of reverse and forward genetics, along with chimeric viruses, to identify viral proteins and residues important for fidelity. These studies will greatly enhance our knowledge of how coronavirus proteins regulate and maintain high-fidelity replication, and will provide valuable insight into the mechanism of ExoN- mediated recognition and repair. This research with likely lead to strategies to broadly combat endemic and emerging coronaviruses, through manipulation of this unique viral RNA proofreading enzyme. Additionally, given the high conservation of nsp14-ExoN within coronaviruses, ExoN inhibitors would represent a novel class of antivirals with potential pan-coronavirus activity.